The present invention relates to reverse transcription of RNA, and in particular to thermostable DNA polymerases that have reverse transcriptase activity.
Many ribonucleic acid (RNA) molecules contain secondary structure that results from hybridization between complementary regions within the RNA molecule. A variety of secondary structures can be formed, including hairpins and cruciforms. RNA molecules containing secondary structure are often difficult to reverse transcribe because polymerases cannot readily process through the secondary structure.
Because of the difficulty of reverse transcribing RNA molecules with secondary structure, many techniques dependent upon reverse transcription yield anomalous results. For example, RNA molecules with secondary structure may be poorly represented in cDNA libraries. Populations of RNA with secondary structure may also yield cDNA libraries with a short insert size. Furthermore, RNA molecules containing secondary structure may be difficult to detect in assays such as reverse transcription-polymerase chain reaction (RT-PCR).
Traditionally, reverse transcription has been performed with reverse transcriptases encoded by retroviruses (e.g., avian myoblastosis virus (AMV) reverse transcriptase and Moloney murine leukemia virus (MMLV) reverse transcriptase). Several mesophillic DNA polymerases (e.g., E. coli DNA polymerase I) have also been shown to possess reverse transcriptase activity. However, these enzymes are generally used at temperatures of between about 37xc2x0 C. to 42xc2x0 C., a temperature range where secondary structure can be a significant problem.
Several thermophilic DNA polymerases (e.g., Thermus aquaticus DNA polymerase and Thermus thermophilus DNA polymerase) also have reverse transcriptase activity. These enzymes are useful for reverse transcription, because at the high temperatures where such enzymes are stable, secondary structure in RNA molecules is reduced. Furthermore, such enzymes can be used to directly synthesize second strand DNA and potentially even to directly amplify an RNA target. However, the utility of these thermostable enzymes is limited because they require manganese as a co-factor for reverse transcriptase activity (e.g., U.S. Pat. No. 5,322,770) resulting in deleterious effects. In some cases, the fidelity of the polymerase is reduced as compared to the fidelity of the enzyme in the presence of other cofactors, such as magnesium ions. Therefore, it is not desirable to amplify the template in the same reaction mixture in which reverse transcription reaction is conducted. This necessitates extra time consuming steps when performing RT-PCR. In other cases, the presence of manganese ions may also cause degradation of the RNA template.
Accordingly, what is needed in the art are alternative thermostable polymerases that have reverse transcriptase activity. Preferably, such thermostable polymerases should have reverse transcriptase activity in the presence of magnesium so that high-fidelity cDNAs may be obtained and so that both reverse transcription and amplification in RT-PCR reactions may conducted in the same reaction mixture.
The present invention relates to reverse transcription of RNA templates, and in particular to reverse transcription by thermostable DNA polymerases. The present invention is not limited to any particular RNA template. Indeed, a variety of RNA templates are contemplated. Examples of RNA templates include, but are not limited to, mRNA, rRNA, purified RNA, mixtures of mRNA, mixtures of rRNA and mRNA, and purified preparations of these various RNAs.
The present invention is not limited to the use of a particular thermostable DNA polymerase. Indeed, the use of a variety of thermostable DNA polymerases is contemplated. In some embodiments, the thermostable DNA polymerase is selected from Thermoactinomyces vulgaris (Tvu) and Bacillus stearothermophilus (Bst) DNA polymerases. In some embodiments, the thermostable DNA polymerase is purified from natural sources, while in other embodiments, the DNA polymerase is generated by recombinant techniques. In still other embodiments, the thermostable DNA polymerase lacks significant 5xe2x80x2 exonuclease activity. In some embodiments, the Tvu polymerase is encoded by an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO 6, and variants or portions thereof. In other embodiments, the Bst polymerase is encoded by an amino acid sequence selected from SEQ ID NO: 19 and SEQ ID NO: 21, and variants or portions thereof.
In some embodiments, the present invention provides methods for reverse transcribing template RNA (i.e., making cDNA copies of the template RNA). In some embodiments, the method comprises a) providing: i) a polymerase selected from T. vulgaris and B. stearothermophilus DNA polymerases; ii) template RNA; iii) at least one primer; and iv) a reaction buffer comprising magnesium ions; b) combining the polymerase, template RNA, at least one primer, and reaction buffer to form a reaction mixture; and c) reacting said reaction mixture under conditions such that the template RNA is reverse transcribed, producing cDNA. The method is not limited by the order in which the polymerase, template RNA, at least one primer, and reaction buffer are combined. In some embodiments, the reaction buffer is substantially free of manganese ions. In other embodiments, the reacting step is performed at about 50 degrees Celsius to about 80 degrees Celsius, preferably at about 60 degrees Celsius to about 75 degrees Celsius. The method is not limited to a particular type of primer. Indeed a variety of primers may be used, including, but not limited to, oligonucleotides complementary to the 5xe2x80x2 untranslated region of an mRNA, the coding region of an mRNA, or the 3xe2x80x2 untranslated region of an mRNA, oligo(dT), and random primers (e.g., random hexamers or octamers). In still further embodiments, the method comprises the additional step d) amplifying the cDNA produced by the reverse transcription reaction.
The present invention also provides methods for detecting the presence of an RNA molecule in a sample by reverse transcription PCR (RT-PCR). In some embodiments, the reverse transcription and amplification reactions are conducted in the same reaction buffer (i.e., a single pot reaction is performed). In other embodiments, reverse transciption and amplification are performed in separate reactions (i.e., a two pot reaction is performed). Accordingly, in some embodiments, the method comprises: a) providing: i) a polymerase selected from T. vulgaris and B. stearothermophilus DNA polymerases; ii) a sample suspected of containing a target RNA; iii) at least a first primer and a second primer, wherein the first primer is complementary to the target RNA and the second primer is complementary to a cDNA copy of the target RNA; and iv) a reaction buffer comprising magnesium ions; b) mixing the polymerase, target RNA, reaction buffers, and primers to form a reaction mixture; c) reacting the reaction mixture under conditions such that the polymerase reverse transcribes the target RNA to produce first strand DNA; and d) reacting the first strand DNA with the second primer under conditions such that second strand DNA is produced; and e) reacting the first strand DNA, second strand DNA, first primer, and second primer under conditions such that a DNA molecule comprising a third strand and a fourth strand is produced, the third strand having a region of complementarity to the first strand and the fourth strand having a region of complementarity to the second strand. In some embodiments, the reaction mixture further comprises an additional thermostable polymerase (e.g., Taq DNA polymerase, Tne DNA polymerase, Pfu DNA polymerase, and the like). In some embodiments, the conditions further comprise heating the reaction mixture. In other embodiments, the conditions further comprise cooling the mixture to a temperature at which the thermostable DNA polymerase can conduct primer extension. In still further embodiments, the heating and cooling is repeated one or more times.
In still further embodiments, the present invention provides mixtures and kits for performing reverse transcription. In some embodiments, the mixture comprises a polymerase selected from T. vulgaris and B. stearothermophilus DNA polymerases, purified RNA, and magnesium ions. In other embodiments, the concentration of magnesium is from about 0.1 to 10 mM, preferably 1 to 5 mM. In another embodiment, the mixture further comprises a buffer. In still other embodiments, the mixture comprises a surfactant. In further embodiments, the mixture has a pH of about 6 to 10, preferably a pH of about 7 to 9. In some embodiments, the reaction mixture further comprises an additional thermostable polymerase (e.g., Taq DNA polymerase, Tne DNA polymerase, Pfu DNA polymerase, and the like)
In other embodiments of the present invention, a kit is provided. In some embodiments, the kit comprises a polymerase selected from T. vulgaris and B. stearothermophilus DNA polymerases, purified RNA as a control template, and a buffer comprising magnesium ions. In some embodiments, the kit contains instructions for performing reverse transcription. In other embodiments, the buffer further comprises a surfactant. In some embodiments, the pH of the buffer is from about 6 to 10, preferably a pH of about 7 to 9. In some embodiments, the kit comprises an additional thermostable polymerase (e.g., Taq DNA polymerase, Tne DNA polymerase, Pfu DNA polymerase, and the like).
The present invention also provides methods for amplifying a double stranded DNA molecule, comprising the steps of: a) providing: i) a first DNA molecule comprising a first strand and a second strand, wherein the first and second strands are complementary to one another; ii) a first primer and a second primer, wherein the first primer is complementary to the first DNA strand, and the second primer is complementary to the second DNA strand; and iii) a first thermostable DNA polymerase derived from T. vulgaris; and b) mixing the first DNA molecule, first primer, second primer, and polymerase to form a reaction mixture under conditions such that a second DNA molecule comprising a third strand and a fourth strand are synthesized, with the third strand having a region complementary to the first strand and the fourth strand having a region complementary to the second strand. In some embodiments, the reaction mixture further comprises an additional thermostable polymerase (e.g., Taq DNA polymerase, Tne DNA polymerase, Pfu DNA polymerase, and the like). The method of the present invention is not limited by the source of the first DNA molecule. In a preferred embodiment, the first DNA molecule is present in a genomic DNA mixture (e.g., in genomic DNA extracted from an organism, tissue or cell line). In alternative embodiments, the first DNA molecule is derived from an RNA molecule using reverse transcriptase-PCR (RT-PCR). The newly synthesized DNA molecule (cDNA) then serves as substrate in the subsequent amplification reaction. The conditions that permit the primer to hybridize to the DNA molecule, and allow the DNA polymerase to conduct primer extension may comprise the use of a buffer.
In one embodiment, the method comprises heating the mixture. In an alternative embodiment, the method further comprises cooling the mixture to a temperature at which the thermostable DNA polymerase can conduct primer extension. In a particularly preferred embodiment, the method comprises repeating the heating and cooling steps one or more times.
It is also contemplated that the polymerase of the method will have various useful properties. It is therefore contemplated that in one embodiment of the method, the Tvu polymerase lacks significant 5xe2x80x2-3xe2x80x2 exonuclease activity. In other embodiments, the polymerase has reverse transcriptase activity in the presence of either magnesium or manganese ions. In still other embodiments, the reverse transcriptase activity in presence of magnesium ions is substantially manganese ion independent.